Region 10 presentation on leveraged research Mar 2006

EPA Leveraged and In-Kind Projects Marc Mills EPA/ORD/NRMRL EPA ORD Performers Marc Mills, Ph.D. USEPA ORD NRMRL, Cincinnati, OH ƒ Contaminant fate and transport in sediments ƒ Porewater characterization H. Joel Allen, Ph.D. ƒ Behavioral toxicology ƒ Watershed protection USEPA ORD NRMRL, Cincinnati, OH Jim Lazorchak, Ph.D. USEPA ORD NERL, Cincinnati, OH ƒ Biological indicators of exposure to contaminated sediments John Meier, Ph.D. ƒ Genotoxicity assays USEPA ORD NERL, Cincinnati, OH Iris Knoebl, Ph.D. USEPA ORD NERL, Cincinnati, OH ƒ Gene expression as an indication of contaminant exposure 1 EPA ORD project focus • Two sites ¾ ¾ Wyckoff/Eagle Harbor Second site to be identified • Focus on biological, physical, and chemical measures of contaminant migration ¾ Porewater characterization • Porewater measures • Conservative tracer ¾ Biological measures of exposure using deployed bivalves • Genotoxicity • Gene expression • Behavioral measures • Bioaccumulation EPA In-kind and Leveraged projects • Pore water characterization • Objectives: • Utilize innovative methods of porewater characterization • Evaluate the transport of porewater using conservative tracer • Performers: Marc Mills, Eric Kleiner • Biological indicators • Objectives: • Provide biological indicators of exposure through capped sediments » Deployed biology » Genetox » Genetic expression • Performers: Joel Allen, John Meier, Iris Knoebl, and Jim Lazorchak 2 Characterizing GW/SW interaction in sediment cap at EH • Goal: Evaluate the tools to characterize the advective flux of GW through the sediment cap • Objective: Measure the head potential in the cap and the underlying sediments ¾ Measure the conductivity of porewater in the cap and the underlying sediments ¾ Measure the temperature of the porewater in the cap and the underlying sediments ¾ Tools to characterize the advective flux of contaminant • SERDP project techniques – Water movement Piezometers ¾ Flux chambers ¾ Hydrological modeling ¾ • EPA project techniques – Porewater characterization Contaminant in porewater ¾ Chemical tracers ¾ 3 EPA – Porewater characterization • Tasks ¾ ¾ EC survey to identify vertical porewater flows – “freshening” Collection and analysis of porewater samples using innovative techniques • Includes: • Porewater points • SPMD spikes • SPME direct sediment measurements ¾ Conservative tracer study • Provides an added piece to the SERDP project which measures advective flux of water but does not measure contaminant flux explicitly Freshening survey • Objective: Map locations of GW movement through the cap • Method: Insert EC probe incrementally into the cap ¾ ¾ ¾ Conductivity Probe: GeoProbe Array SC-300 w/ Top Dipole Surveyed on transects on 10 m intervals Typical Tidal Pattern in Eagle Harbor (Source = NOAA) Dive support from EPA Reg 10 Dive team 14 12 10 Harbor Level (ft) • Initial survey in May 2005 8 6 4 2 0 0 -2 Time (hrs) 24 48 72 4 Equipment for Conductivity Survey To surface for instrument reading Drive cap/pull cap 3 ft Drive rod (1/2” dia. tempered steel rod) Increments for profile (cable ties set at 10cm increments) Drive rod (1/2” dia. tempered steel rod) 2 ft Conductivity probe GeoProbe Array SC-300 w/ Top Dipole Conductivity survey results – May 2005 5 EC survey – 0.3 m above sediment EC survey – 0.3 m below sediment 6 EC survey – 0.8 m above sediment Vertical profile of EC in cap (Transect 1) 7 Three dimensional representation of EC survey data Conductivity survey results and conclusions • Results Areas of GW freshening through the cap were identified for subsequent study ¾ Areas with little or no GW freshening were also identified for subsequent study ¾ • Conclusions Notable that survey occurred in May 2005 during a very low rainfall period for the area – may therefore under-represent a more typical condition ¾ Will be used along with contaminant data, cap thickness, and other parameters to identify candidate locations for the research ¾ Prior to deploying further equipment on/in the cap, EC will be confirmed with additional measures ¾ 8 Sediment piezometer deployment Sediment porewater sampler Well screen Sample dip tube 9 Sediment porewater sampler SPMD Sediment “spike” 10 Option 1: Buoy based P B B TG P P P UW Sediment sampling point • Design allows for purging the surface water from the point after installation Umbilical (vent and purge llineto the surface to allow the purging This design will also allow for sampling porewater ¾ • • ¾ Inert materials – PAHs and Tracers Non-inert materials – Tracer only • This design makes maintenance or troubleshooting much more difficult (have to pull sonde to service probes) 11 Sediment mini-wells Water Cap • Design allows for purging the surface water from the point after installation • Umbilical (vent and purge llineto the surface to allow the purging • This design will also allow for sampling porewater ¾ ¾ Inert materials – PAHs and Tracers Non-inert materials – Tracer only • This design makes maintenance/troubleshooting easier but still requires dive support. • Dead volume issues! Sediment EPA – Biological indicators • Deployed biology ƒ ƒ ƒ Mytilus trussulus Shellfish transfer permits being completed Russell Rogers and David Gadwa - WA Dept. of Fish and Wildlife Assays ƒ ƒ ƒ ƒ Gene expression assays to indicate exposure Comet Assays for indication of PAH induced genetic damage Bivalve monitors for behavioral Uptake or body burden ƒ Deployed in two configurations ƒ Caged (gene expression, genotox, and uptake) ƒ Bivalve monitors (behavioral) ƒ Collection and analysis of indigenous indicator species • Provides an additional piece to the SERDP project that focused on physical and chemical measures 12 Gene Expression assays to indicate exposure– Development and optimization of Q-PCR assay to test for PAH exposure in mussels • Literature shows that expression of the CYP4 gene (gene for cytochrome P450 enzyme involved in metabolism of toxins) is affected by exposure to PAHs • A Quantitative PCR assay (Q-PCR) can measure expression of a gene of interest if the gene sequence is known. STEP 1 - Isolate and sequence CYP4 gene from mussels Expose mussels to creosote in the lab Remove digestive gland and store in buffer Isolate RNA Convert RNA to cDNA bp PCR Ladder Product 2000 1200 Perform PCR using degenerate primers for CYP4 gene 800 400 PCR product Containing CYP4 gene 200 13 STEP 2 • Use sequence to obtain primers for Q-PCR • Optimize and test Q-PCR assay using laboratory exposed mussels STEP 3 • Deploy mussels at test site and collect after X days • Use Q-PCR assay to measure CYP4 gene expression to test for PAH exposure Genotoxicity as an indicator of exposure Desirable features of Comet Assay for Measuring DNA damage Sensitive to various types of DNA damage (doublestrand and single-strand breaks as well as alkaline labile sites, e.g. DNA adducts). ¾ Applicable to any tissue and organism of concern, including Mytilus sp. ¾ Small number of cells required for analysis (100 per animal) ¾ Sensitive to detecting PAH exposure in lab and field studies ¾ 14 Comet Assay Procedure for Measuring DNA Damge in Mussels 1. Withdraw hemolymph from posterior adductor muscle with syringe & needle. 2. Centrifuge, resuspend pellet (hemocytes) in agarose, and spead onto slide. 3. Lysis overnight in SDS/high salt to prepare nuclei. 4. DNA unwinding in alkali solution. 5. Electophoresis 6. Neutralize and stain with ethidium bromide 7. Analyze cells by image analysis for extent and amount of migration of DNA into “comet tail” 15 Harvesting the hemolymph DNA Damage in Erythrocytes of Brown Bullheads from a Contaminated Site (Ashtabula R.) and Reference Site (Conneaut R.) 60 50 40 30 20 10 0 Tail Length (µm) Tail DNA (%) Tail Extent Olive Tail Moment (µm) Moment (µm) 23 23 24 23 24 23 24 Ashtabula 24 Conneaut 16 Deployed biology • Bivalve monitors for behavioral studies ¾ ¾ Instrumentation allows monitoring of the behavior of the bivalve Compare the behavior of the exposed to the control Deploy mussels for PAH uptake Recover mussels distribute to: • Uptake • Gene expression • Genotox • Body burden or uptake study ¾ ¾ Bivalve monitor instrumentation 17 18 Mean Gape of 15 Bivalves Exposed to 250μg/L Cu Mean Gape 0 50 100 03:00 07:00 11:00 Time(hours) 15:00 19:00 23:00 19 Questions? Logistics • Q/A ¾ Sediment deployment layouts • • • • Phone lines Power lines Security for dataloggers Telemetry or Hardwire ¾ Benchmarks • Where are they? ¾ Buoys • USACE support • OK on cap? 20 Option 1: Buoy based Option 1: Bouy Based, buoy vented Power Radio signal are provided by solar datalogger/RF station 1a - The vented sondes cables are brought to the surface and vented and “ganged” into common comm, cables which are layed back down on the cap surface and routed to a datalogger/radio on a central located buoy. 1b – Vented sonde cable are run along the bottom to a single buoy. The cables are brought to surface and connected to a central datalogger radio on a centrally located buoy. P B B TG P P P UW Base Station is located at the Upland well and will receive signal from the buoy and then use cell phone for transmitting data. Option 1: Bouy Based, buoy vented Datalogger/Base station SP powers the DL and the LTC sondes Cell modem for telemetry Datalogger/radio SP powers the DL and the LTC sondes/tide gauge Power and Radio signal are provided by solar datalogger/RF station 1b - Vented sonde cable are run along the bottom to a single buoy. The cables are brought to surface and connected to a central datalogger radio on a centrally located buoy. Base Station is located at the Upland well and will receive signal from the buoy and then use cell phone for transmitting data. Buoy tether (cable or chain) Vented Comm line for LTC sonde Common comm line (not vented) Porewater deployments Not to scale 21 Option 2: Land Based, buoy vented The sondes cables are brought to the surface and vented and “ganged” into common communication cables which are layed back down on the cap surface and routed to a secure datalogger within the fence line. P B B TG P 2a – Phone and Power are hard wired to the Site 2b – Phone and Power are provided by solar panel/cell modem P P B TW Option 2: Land Based, buoy vented The sondes cables are brought to the surface and vented and “ganged” into common communication cables which are layed back down on the cap surface and routed to a secure datalogger within the fence line. 2a – Phone and Power are hard wired to the Site 2b – Phone and Power are provided by solar panel/cell modem Buoy tether (cable or chain) Vented Comm line for LTC sonde Common comm line (no vented) “Gang” box to vent LTC cables/comm Porewater deployments Not to scale 22 Option 3: Land Based, land vented The sondes cables are brought to the surface and vented and “ganged” into common communication cables which are layed back down on the cap surface and routed to a secure datalogger within the fence line. TG P B B 3a – Phone and Power are hard wired to the Site 3b – Phone and Power are provided by solar panel/cell modem P P P B TW Option 3: Bouy Based, buoy vented Power Radio signal are provided by solar datalogger/RF station Vented sonde cable are run along the bottom to a central datalogger/base station on a centrally located within the fence line. I would wrap all the lines as they come together with dive support to avoid some many loose lines. Buoy tether (cable or chain) Vented Comm line for LTC sonde Common comm line (not vented) Porewater deployments Not to scale 23